Our working hypothesis is that the local control of tissue blood flow reflects the coordination of activity among endothelial cells and smooth nnuscle cells of microvascular resistance networks. The Specific Aims of this project focus on understanding how rapid (electrical) and slow (calcium waves) components of conducted vasodilation are initiated, how they travel from cell to cell, and how they interact with each other. In accord with the long term nature and innovative scope of this MERIT Award, we have invested substantively in developing novel methods to study these relationships. In freshly isolated endothelial tubes of feed arteries from mouse skeletal muscle, dual simultaneous intracellular recording demonstrates that electrical conduction along the endothelium through gap junctions can be tuned by governing charge loss through potassium channels in the plasma membrane. Complementary experiments show that glycyrrhetinic acid derivatives used widely by others to inhibit gap junctions concomitantly block potassium channels, disqualifying these agents in resolving respective determinants of electrical signaling. We quantified calcium signaling in endothelial tubes using Fura-2 photometry, establishing a foundation for implementing confocal imaging and multi-photon technology which we are using to investigate mechansims of calcium signaling within and between individual cells along the endothelium. In developing intravital macrozoom imaging of transgenic mice that express the calcium-sensitive protein GCaMP2 in arteriolar endothelium, we determined that the initiation of calcium waves with acetylcholine microiontophoresis was independent of myogenic tone and distinguished between the local initation of conducted responses from more global effects when the agonist gained access to the flow stream. In light of studies of myoendothelial coupling that have been based entirely upon isolated preparations studied in vitro, we are investigating endothelial calcium signaling in response to neural activation of smooth muscle cells in vivo. We plan to continue pursuing the Specific Aims of this project in light of our new methodology and findings. Our long-term goals center on defining the signaling events which coordinate the activity of endothelium and smooth muscle in resistance vessels that control the delivery of oxygen and nutrients to tissue cells. Resolving these relationships will provide critical new insight for determining how respective signaling pathways may be affected (and thereby treated) during conditions known to alter endothelial and vascular smooth muscle function including widespread diseased states of obesity, diabetes and hypertension.